Research ArticleTherapeutic Effects of the Combination of Alpha-Lipoic Acid(ALA) and Coenzyme Q10 (CoQ10) onCisplatin-Induced Nephrotoxicity

Eman Aly Khalifa,1 Ahmed Nabil Ahmed ,1,2 Khalid Shaaban Hashem,3

and Ahmad Gad Allah4

1Department of Biotechnology and Life Sciences, Faculty of Postgraduate Studies for Advanced Sciences, Beni-Suef University,Beni-Suef, Egypt2International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS),Tsukuba, Japan3Department of Biochemistry, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt4Department of Physiology, Faculty of Medicine, Al Azhar University, Assiut, Egypt

Correspondence should be addressed to Ahmed Nabil Ahmed; drnabil_100@psas.bsu.edu.eg

Received 16 January 2020; Revised 30 January 2020; Accepted 16 March 2020; Published 9 April 2020

Academic Editor: Istvan Boldogh

Copyright © 2020 Eman Aly Khalifa et al. *is is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Background. Nephrotoxicity of cisplatin has been recognized since its introduction more than 25 years ago. However, despiteintense efforts to develop less toxic and equally effective alternatives, cisplatin continues to be widely prescribed. Aim andObjectives. *e study is aimed at assessing the possible prophylactic effect of coenzyme Q10 (CoQ10) and alpha-lipoic acid (ALA)(separately or in combination) on experimentally cisplatin-induced nephrotoxicity. Subjects and Methods. An experimental studywas performed on adult male albino rats (n� 40), weighing 200–250 g. Rats were randomly divided into 5 groups: group I (normalsaline control), group II (cisplatin control), group III (CoQ10 and cisplatin), group IV (ALA and cisplatin), and group V (CoQ10,ALA, and cisplatin). CoQ10 and/or ALA were given as pretreatment for 9 days, followed by cisplatin injection in the 10th day ofthe study, followed by a short posttreatment course for 3 days. Renal functions, tissue antioxidant activity, and inflammatorymarkers (tumor necrosis factor, TNF) were estimated along with histopathological study. Results. Renal function tests and urinaryproteins were significantly higher within group II compared with other groups (P value <0.001). Creatinine clearance wassignificantly higher with combination therapy (group V compared to other groups). Both TNF andmalondialdehyde (MDA) weresignificantly higher within group II whereas GSH content, catalase, and superoxide dismutase (SOD) were significantly lower ingroup II. MDA level was significantly lower when combination therapy was used. Marked renal damage was histologicallydetected in the cisplatin group, whereas the least renal damage was noticed in the combination group. Conclusion. *e studyconfirmed the role of antioxidants in preventing nephrotoxicity caused by cisplatin; the prophylactic effect of combined therapywith CoQ10 and ALA is superior to that of monotherapy.

1. Introduction

Cisplatin (cis-diamminedichloroplatinum [II], CDDP) is themost significant platinum anticancer medication that isgenerally used to treat a variety of tumors including head,neck, and lung malignancies [1]. Reduction ratesapproached >90% in testicular malignancy [2]. However,

significant side effects were reported by the administrationof high doses of cisplatin. *e major side effects includeneurotoxicity, ototoxicity, and nephrotoxicity [3]. It wasfound that renal dysfunction occurs in more than 70% ofpediatric patients treated with cisplatin [4].

Numerous processes and mechanisms lead to cisplatinnephrotoxicity and contribute to its complexity. However,

HindawiInternational Journal of InflammationVolume 2020, Article ID 5369797, 11 pageshttps://doi.org/10.1155/2020/5369797

the generation of toxic reactive oxygen species (ROS) re-mains a significant causative agent [3]. An importantcontributor for cisplatin nephrotoxicity is its take-up intokidney cells. *e uptake of cisplatin by kidney cells is muchhigher than any tissues, especially in proximal tubules of thekidney where cisplatin concentration can reach up to fivefolds higher than that of the serum [5]. Cisplatin additionallyrepresses the antioxidant enzymes including glutathioneS-transferase, glutathione peroxidase, and superoxide dis-mutase, prompting lethal degrees of ROS inside the cell [6].*e resulting ROS suppresses the respiratory chain and ATPgeneration. *is leads to disturbance of the function of cellsand destruction of cell proteins, lipids, and nucleic acids;prompts endoplasmic reticulum stress; and causes cell ne-crosis [7]. Other mechanisms that may play a role innephrotoxicity by cisplatin include inflammation [8] and cellapoptosis [9].

Several lines of evidence suggest that mitochondrialDNA or other mitochondrial targets are perhaps moreimportant than nuclear DNA damage in mediating cis-platin-induced cell death. Cisplatin is hydrolyzed to generatea positively charged metabolite which preferentially accu-mulates within the negatively charged mitochondria. *us,the sensitivity of cells to cisplatin appears to correlate withboth the density of mitochondria and the mitochondrialmembrane potential. *is observation may explain theparticular sensitivity of the renal proximal tubule to cisplatintoxicity, as this segment exhibits one of the highest densitiesof mitochondria in the kidney [10].

An antioxidant is defined as any substance that, whenpresent at low concentrations, delays or prevents oxidationsof cell components like lipids, proteins, carbohydrates, andDNA. Superoxide dismutase (SOD), catalase (CAT), andglutathione reductase represent the first line antioxidantswithin the human body [11].

*ere are two important metabolic oxidants in themitochondria, namely, coenzyme Q (CoQ10) and lipoicacid. *e presence of these molecules shows that mito-chondria as individual organisms can defend themselvesagainst the harmful effects of the oxygen atmosphere [12].

Alpha-lipoic acid (ALA) is derived from octanoic acid.In the mitochondria, it acts as a cofactor of mitochondrialα-ketoacid dehydrogenase [13]. ALA increases the level ofantioxidant enzymes and aids in recycling of vitamins E andC. Because of these properties, ALA is known as the“universal antioxidant.” Besides, ALA has anti-inflamma-tory action apart from its antioxidant activity. *e anti-inflammatory effects of LA are due to suppression of in-flammatory activity of NF-kB, TNF-a, and IL-6 and in-creased anti-inflammatory proteins, such as nuclearerythroid 2-related factor (Nrf2) [14].

In vivo lipoic acid is easily converted into its reducedform, dihydrolipoic acid (DHLA). For a long time, ALA hasbeen known as a cofactor of a-ketoacid dehydrogenases, butrecent research has been focused mainly on the antioxidativeproperties of ALA and DHLA. *ese two compounds havebeen reported as effective free radical scavengers and metalchelators acting in vitro as well as in vivo. DHLA can re-generate reduced forms of essential intracellular

antioxidants such as ascorbate and tocopherol. Moreover,ALA has been reported to affect the activities of transcriptionfactors, NF-kB and AP-1 [15].

Furthermore, ALA has the ability to chelate toxic metalsin a direct way or through its ability to increase glutathionelevels inside the cells. Because of these significant antioxi-dant activities, ALA is used to protect against oxidativedamage in various diseases such as diabetes, neuropathy,cataracts, and aging [16–18].

Coenzyme Q10 (CoQ10) is a lipid-soluble vitamin-likesubstance, also known as ubiquinone. CoQ10 is similar tovitamin K in chemical composition; however, it is notconsidered as a vitamin because it can be synthesized insidethe body. CoQ10 is found in the membranes of cell or-ganelles, especially the inner mitochondrial membrane. It isthe single naturally occurring lipid soluble antioxidant thatcan be synthesized endogenously. It participates in aerobiccellular respiration working as an electron carrier in theprocess creating energy through the formation of ATP(adenosine triphosphate) [19–21]. CoQ10 has been dem-onstrated to be a regulator of vasoactive reactions; thus it wasextensively studied in prevention and treatment of diabetes,cardiovascular, and renal disease [12].

For several years, coenzyme Q (CoQ10 in humans) wasknown for its key role in mitochondrial bioenergetics; laterstudies demonstrated its presence in other subcellularfractions and plasma and extensively investigated its anti-oxidant role. *ese constitute the basis on which researchsupporting the clinical use of CoQ10 is founded. Also, at theinner mitochondrial membrane level, coenzyme Q is rec-ognized as an obligatory cofactor for the function ofuncoupling proteins and a modulator of the transition pore[19].

In the reduced form as ubiquinol, CoQ10 inhibits theperoxidation of cell membrane lipids and can act as anti-oxidant outside the mitochondrial membrane. Not only canit recycle and regenerate other antioxidants such as vitaminsE and C, but it can also uniquely affect the initiation andpropagation of ROS [22].

Another notable function of CoQ10 would be the abilityto interact with dihydrolipoic acid (DHLA), by transferring apair of electrons. *is helps to keep CoQ10 in the reducedstate, thereby maximizing antioxidant capacity in otherextramitochondrial membranes [23]. *e modulatory effectof CoQ10 on gene expression has also been reported [24].

It was found that antioxidants either work separately orcollaborate when they are oxidized [16]. *e generation offree radicals certainly plays a central role in the developmentof different pathological conditions. Most of these patho-logical conditions are multifactorial in origin. Hence, the useof a combination of antioxidants, rather than antioxidantmonotherapy, is strongly recommended [12].

*e abovementioned data suggests that ALA and CoQ10share the following common properties that may facilitatetheir synergistic activity. First, CoQ10 can interact withDHLA to remain in the reduced state. Second, both work asdirect antioxidants and ROS scavengers.*ird, both increasethe activity of glutathione and other antioxidant enzymes.Fourth, both contribute to the recycling of vitamins E and C.

2 International Journal of Inflammation

Last, ALA and CoQ10 modulate the transcription factorsand gene expression responsible for mitochondrial activityand stress response (Figure 1). For these reasons, the authorshypothesized that this combination may be beneficial inreducing cisplatin nephrotoxicity. *e study was planned toinvestigate the possible prophylactic benefits of combiningCoQ10 and ALA as an early treatment against cisplatin-induced nephrotoxicity.

2. Subjects and Methods

2.1. Technical Design. *e present study has been completedin biochemistry labs at Beni-Suef University, Egypt.

2.2. Inclusion Criteria. *is study has been performed onadult male albino rats (Wistar strain) (n� 40), weighing200–250 g. Rats, 8–10 weeks of age, were housed in acontrolled situation with a 12 h light/dark cycle, at 23± 2°C,50± 5% relative humidity. Precautions were implemented toavoid exposure of rats to any type of stress.

2.3. Animal Groups. Rats had been permitted to adapt forabout fourteen days before the experimentation, and af-terward they were haphazardly separated into five groupsand permitted to have free access to an ordinary laboratoryrat diet and tap water. Each group contained 8 rodents;groups were classified as follows:

Group I (normal saline group): normal saline 0.5mlwas given by intraperitoneal infusion (IP) on the tenthday of the trial.Group II (cisplatin control): cisplatin (6mg/kg) inisotonic saline was given by intraperitoneal infusion(IP) on the tenth day of the trial.Group III (coenzyme Q 10 group): a daily portion ofCoQ10 (100mg/kg) was given orally for 13 days, andcisplatin (6mg/kg) was given (IP) on the tenth day ofthe test.Group IV (lipoic acid group): ALA (100mg/kg) wasgiven orally for 13 days, and cisplatin 6mg/kg (IP) onthe tenth day of the test.Group V (combination group): this group was givenboth ALA (100mg/kg) daily and CoQ10 (100mg/kg)orally for 13 days. Cisplatin (6mg/kg) was given (IP) onthe tenth day of the test.

2.4. Operational Design. Doses and timing were chosenbased on other related researches [25–27]. Animals had beensacrificed 72 hrs after cisplatin or saline injection, and thenblood and kidney tissue samples were collected.

2.4.1. Chemicals and Drugs. Cisplatin vials (Bristol MyersSquibb Co., USA) were utilized. DL-α-lipoic acid was pur-chased from Sigma-Aldrich Chemie, Germany. CoQ10(100) mg was purchased from California Gold Nutrition®,USA. Every single other compound and reagents utilized were

of the most noteworthy virtue grade accessible. CoQ10 sus-pension was prepared in 0.9% of normal saline and admin-istered orally to group III (alone) and group v (combined withALA) for 13 days. Lipoic acid group (group IV) was given adaily dose of lipoic acid (100mg/kg) orally for 13 days; it wasalso given to group V (the combination group).

2.4.2. Animals. Adult male Wistar albino rats weighing200–250 g were purchased from the Egyptian Organization forBiological Products and Vaccines (VACSERA, Giza, Egypt).

2.5. Preparation of the Kidneys. Both kidneys were rapidlyexcised and weighed. *e renal cortex was then separatedand kept at −8°C. *en it was homogenized using coldpotassium phosphate buffer (0.05 M, pH 7.4), followed bycentrifugation for obtaining the supernatant. Kidneys werethen cut longitudinally, followed by fixation in 10% for-malin, and then they were embedded in paraffin for his-topathological examination.

2.6. Renal Homogenate. Renal homogenate was utilized forassessment of total protein level and antioxidant parameterswithin the kidney; these include renal tissue glutathione(GSH), superoxide dismutase (SOD), and catalase activities.

2.6.1. Measurement of Tissue Glutathione (GSH) Content.Reduced GSH was estimated spectrophotometrically at412 nm as indicated by the technique for Ellman [28]. T-GSHassay kit E-BC-K097-M, Elabscience®, USA, was used. To eachmicrocentrifuge tube containing 0.5ml TCA-EDTA, 0.5ml ofdiluted samples was added. *e tubes were gently shakenintermittently for 15 minutes, followed by centrifugation at2000 rpm at room temperature for 5minutes. An aliquot 0.1 ofthe resultant clear supernatant was aspirated and mixed with1.7ml phosphate buffer in separate test tubes. A duplicate wasmade for each sample, and then 0.1ml Ellman’s reagent wasadded to each tube. After 5min, the optical density wasmeasured at 412 nm against a blank using a spectropho-tometer. A set of serial dilutions of reduced GSH were used toconstruct a calibration curve. *e measure of GSH wasexpressed as μmol/g tissue.

2.6.2. Measurement of Superoxide Dismutase (SOD) Activity.SOD activity was determined depending on the inhibition ofpyrogallol oxidation by SOD [29]. Pyrogallol solution wasobtained from Sigma-Aldrich (product number 254002).*e other chemicals used in the test were purchased locally.An aliquot of diluted sample (100 μl) was added to 25 μl ofpyrogallol (24mmol/L prepared in HCl), and the finalvolume was adjusted to 3ml using Tris HCl (0.1 M, pH 7.8).*e changes in the absorbance at 420 nmwere recorded for 3minutes at 1min interval, using a spectrophotometer. Oneunit of SOD activity is defined as the amount of the enzymecausing 50% inhibition of autooxidation of pyrogallol. SODactivity was expressed as U/mg protein (or units/min/g wettissue).

International Journal of Inflammation 3

2.6.3. Measurement of Catalase Activity. *is was estimateddepending on the decrease in absorbance at 240 nm due todecomposition of hydrogen peroxide by catalase [30].Catalase (CAT) assay kit E-BC-K031-S, Elabscience®, USA,was used. In a 3ml quartz cuvette, 100 μl of homogenate(10%) was added to 2.9ml of 19mmol/L H2O2 solutionprepared in potassium phosphate buffer (0.1 M, pH 7.4).*ereaction was monitored by continuous recording of thechange in the absorbance at 240 nm every minute for 2minusing Jenway 6305 UV/visible spectrophotometer, expressedas U/g wet tissue.

2.7. Assessment of Renal Lipid Peroxidation. *is was eval-uated by measuring malondialdehyde (MDA) in tissueswhich is broadly acknowledged as a marker of lipid per-oxidation. MDA is formed from the breakdown of

polyunsaturated fatty acids and serves as a convenient indexfor determining the extent of the peroxidation reaction.MDA was measured based on colourimetric determinationof thiobarbituric acid reactive substance (TBARS) [31].MDA assay kit (ab118970) was purchased from Abcamdistributed by GeneTECH (Egypt) (TBARS). Determinationof MDA is based on the reaction of one molecule of MDAwith two molecules of thiobarbituric acid at low pH (2-3).An aliquot of 0.5ml of diluted sample was pipetted into a10ml centrifuge tube, followed by the addition of 3ml of 1%orthophosphoric acid and 1ml of 0.6% thiobarbituric acid.*e tubes were incubated in a water bath at 95°C for 45min.After cooling the mixture, 4ml of n-butanol was added toeach tube and mixed vigorously. *e butanol phase (upperlayer) was separated by centrifugation at 2000 rpm for10min. *e color was measured at 535 and 520 nm using aspectrophotometer. *e difference in optical density

Tocopheroxylradical

Dehydro ascorbate radical

Ascorbate (VitaminC)

Alpha-tocopherol(Vitamin E)

Glutathionedisulphide GSSD

Glutathione (GSH)

Ubiquinol(CoQH)

Semiquinone radical

Lipoic acid (LA)

Dihydrolipoic acid

DHLA

NADP

NADPH

ROO

ROOH

Modutation of theexpression of

transcription factors/induction of antioxidant

genes

Increaesed mitochondtialactivity, strss response,increased glutathione

Metalchelator

Anti-inflammatory

Figure 1: Interactions between CoQ10, ALA, and other antioxidants.

4 International Journal of Inflammation

between both wave lengths was used as a measure of MDAabsorbance. A calibration curve was constructed using serialdilutions of 1,1′,3,3′-tetramethoxypropane. *e results wereexpressed as nmol/g tissue.

2.8. Quantitative Analysis of Tumor Necrosis Factor-α (TNF-α). *is was done by ELISA quantitative enzyme immu-noassay technique, Quantikine® ELISA, USA (TNF-α is acisplatin-induced inflammatory cytokine). A monoclonalantibody specific for rat TNF-α was precoated onto amicroplate. Standards, control, and samples were pipettedinto the wells, and any TNF-α present was bound by theimmobilized antibody. After washing away any unboundsubstances, an enzyme-linked polyclonal antibody specificfor rat TNF-α was added to the wells. Following a wash toremove any unbound antibody-enzyme reagent, a substratesolution was added to the wells.*e enzyme reaction yields ablue product that turns yellow when the stop solution wasadded. *e intensity of the color measured was in pro-portion to the amount of TNF-α bound in the initial step.*e sample values were then read off from the standardcurve.

2.9. Histopathological Examination. Kidney tissues werekept in 10% formalin, and dehydration was done by alcohols,followed by clearance by xylene and then insertion inparaffin wax. Sections were then stained using hematoxylinand eosin. *e amplification intensity of 400 was utilized todemonstrate the histopathological changes utilizing lightmicroscopy.

2.10. Biochemical Examination of Blood and Urine Samples

Blood samples were used to measure serum creatinine,blood urea, and serum uric acid. Reagents were ob-tained from BIOMED diagnostics, Egypt: urea(URE118100), uric acid (UA119090), and creatinine(CRE 106100).Urine samples were utilized for determination of uri-nary albumin and total protein. *e urinary creatininewas estimated to measure creatinine clearance. Re-agents were obtained fromDiamond Diagnostics, USA,distributed by Lab Supply, Egypt: albumin (BK-467858D) and total protein (BK-465986D).

2.11. Protocol Approval by the Ethical Committee. Before theestablishment of the study, based on local regulation, thestudy protocol was submitted and approved by the Ethicaland Research Committee (Beni-Suef University InstitutionalReview Broad (IRB)).

2.12. Data Management and Statistical Analysis. Data pro-cessing and statistical analysis were done using MedCalc ver.18.2.1 (MedCalc, Ostend, Belgium). Kruskal–Wallis one-way ANOVA test was the main test used in the study. It wasused to compare nominal variables among three or moregroups. Other less frequently used tests includedWilcoxon’s

test that was used to compare the control group with any ofthe other groups; Chi-square and logistic regression analysiswere used for categorical variables. Spearman’s correlationwas used for continuous and ordinal variables. *e last threetests were used mainly in an attempt to relate the histo-pathological findings to the treatment groups. P values lessthan 0.05% were considered to be statistically significant,and less than 0.001 highly significant.

3. Results

3.1. Biochemistry Results. One mortality was reported ingroup II (48 hours after cisplatin injection), with no reportedmortality among other groups. Blood urea, serum creatinine,serum uric acid, urinary albumin, and total protein levelswere significantly higher within group II compared withgroup V, group IV, and group III (P value <0.001); these areshown in Table 1. No significant difference was found amongthe groups treated with ALA and/or CoQ10 regarding theseparameters (groups III, IV, and V). Similarly, it was foundthat creatinine clearance was significantly reduced whengroup II was compared to other groups. When combinationtherapy was compared to monotherapy (group V comparedto group III and group IV), it was found that combinationtherapy resulted in a significant elevation of creatinineclearance (P value <0.05), Figure 2.

3.2. Oxidative Stress Markers and Inflammatory Markers.TNF (an inflammatory cytokine) and MDA levels weresignificantly higher within group II compared to groups III,IV, and V. Meanwhile GSH content, catalase, and SOD weresignificantly lower within group II compared to other groups(P value <0.05). Treatment with CoQ10 and/or ALA resultedin a significant reduction in the MDA level caused by cis-platin. Moreover, when group V (combined therapy) wascompared to monotherapy groups (groups III, IV), it wasfound that combined therapy is more effective in reducingMDA level than monotherapy; this difference was statisti-cally significant (P value <0.05) (Table 2, Figure 3).

3.3. Histopathological Findings. A modified pathologicalscoring system was used to subjectively assess 8 morpho-logical parameters; the given scores ranged from 0 to ++[32]. It was noted that marked renal damage occurred incisplatin treated group (group II), and the least renal damagewas noticed in the combination group (group V) (Figure 4).A detailed description was as follows;

Group I (control): kidney showed average renal cap-sule, average glomeruli with average Bowman’s spaces,average proximal tubules with preserved brush borders,and average distal tubules, and renal medulla showedaverage collecting tubules with average interstitium.Group II (cisplatin): kidney showed partially detachedrenal capsule, scattered atrophic glomeruli with wid-ened Bowman’s spaces, proximal tubules with edem-atous, apoptotic and necrotic epithelial lining, completeloss of brush borders, intratubular hyaline casts, and

International Journal of Inflammation 5

markedly dilated congested interstitial blood vessels,and renal medulla showed markedly dilated collectingtubules, with atrophied epithelial lining, and intra-tubular hyaline casts.Group III (cisplatin +CoQ10): kidney showed averagerenal capsule, scattered atrophic glomeruli with wid-ened Bowman’s spaces, proximal tubules with edem-atous and apoptotic epithelial lining and partial loss ofbrush borders, intratubular cellular debris and hyalinecasts, and markedly dilated congested interstitial blood

vessels with areas of hemorrhage, and renal medullashowed collecting tubules with edematous and apo-ptotic epithelial lining, mildly dilated congested bloodvessels, and intratubular hyaline casts.Group IV (cisplatin +ALA): kidney showed averagerenal capsule, scattered small-sized glomeruli withwidened Bowman’s spaces, proximal tubules withedematous and apoptotic epithelial lining, partial lossof brush borders, and mildly dilated congested inter-stitial blood vessels, and renal medulla showed col-lecting tubules with average epithelial lining andintratubular hyaline casts.Group V (cisplatin +CoQ10 +ALA): Kidney showedaverage renal capsule, average glomeruli with averageBowman’s spaces, proximal tubules with edematousand apoptotic epithelial lining, partial loss of brushborders, intratubular hyaline casts, and mildly dilatedcongested interstitial blood vessels, and renal medullashowed collecting tubules with edematous epitheliallining and intratubular cellular debris. Table 3 sum-marizes the histopathological findings in the differentstudied groups.

4. Discussion

Despite the fact that different compounds may be termed‘‘antioxidants,” still vitamin E, vitamin C, coenzyme Q10(CoQ10), and alpha-lipoic acid (ALA) are among the mostextensively studied ones. Cumulative data have proved that

Table 1: Biochemistry data in the studied groups.

Group I(N� 8)

Group II(N� 7)

Group III(N� 8)

Group IV(N� 8)

Group V(N� 8)

P value in groupII (cisplatin)versus other

groups

P value in group V(combination)

versusgroups III, IV(monotherapy)

Urea (mg/dl)Mean± SD 17.36± 4.61 227.7± 9.3 171.13± 6.19 162.8± 5.99 158.38± 6.86 0.00081∗ NS

Urinaryalbumin(mg/24 h)Mean± SD

23.61± 11.3 478.6± 37.6 257.8± 12.1 289.13± 17.2 231± 4.59 0.00093∗ NS

Urinary totalprotein(mg/24 h)Mean± SD

302.32± 62.9 3113.8± 118.4 1503.3± 69.4 1495.9± 106.4 1296.8± 75.4 0.00086∗ NS

Uric acid(mg/dl)Mean± SD

3.7± 0.246 8.33± 0.164 3.92±0.0797 3.99± 0.111 3.69± 0.066 0.00075∗ NS

Creatinine(mg/dl)Mean± SD

1.5± 0.541 6.01± 0.376 3.38± 0.227 3.801± 0.178 2.96± 0.097 0.00065∗ NS

Creatinineclearance(ml/min)Median(range)

0.3610.09–0.83

0.0040.002–0.006

0.03150.028–0.038

0.04050.022–0.054

0.10850.096–0.199 0.00081∗ 0.0176∗

∗Significant; NS: nonsignificant.

0.361

0.004∗0.0315 0.0405

0.1085∗∗

00.05

0.10.15

0.20.25

0.30.35

0.4

Creatinine clearance

GIGIIGIII

GIVGV

Figure 2: Creatinine clearance in the studied groups. Creatinineclearance was significantly reduced when group II was compared toother groups. ∗P value was 0.00081. Combination therapy resultedin a significant elevation of creatinine clearance when compared tomonotherapy (i.e., group V compared to group III and group IV).∗∗P value was 0.0176.

6 International Journal of Inflammation

ALA exhibits significant protective antioxidant effectsagainst oxidative damage encountered in several diseasessuch as diabetes mellitus, neuropathy, renal diseases, andaging [16–18]. CoQ10 has been also shown to be a powerfulantioxidant. Its role in the prevention and treatment ofdiabetes, cardiovascular, and renal diseases was investigatedin different studies [19].

*e protective role of CoQ10 when combined with ALAin prevention of nephrotoxicity was not fully investigated.*is study was an attempt to evaluate the potential syner-gistic activity of CoQ10 and ALA during cisplatin-inducednephrotoxicity.

All antioxidants work in a coordinated system to controllevels of free radical formation, and deficiencies in one el-ement can affect the others. As antioxidants, CoQ10 andALA are interrelated, complementary to each other, andinvolved in the different pathways that generate and recycleother antioxidants, mainly glutathione, vitamin E, and vi-tamin C (Figure 1). Glutathione, an important water-solubleantioxidant, directly quenches ROS such as lipid peroxidesand also plays a major role in the body’s detoxificationprocess. Research suggests that glutathione and vitamin Cwork interactively to quench free radicals and that they havea sparing effect upon each other [33].

In extracellular fluids, vitamin C is considered the mostimportant water-soluble antioxidant. It is capable of neu-tralizing ROS in the aqueous phase before lipid peroxidationis initiated. Vitamin E, a major lipid-soluble antioxidant, isthe most effective chain-breaking antioxidant within the cellmembrane where it protects membrane fatty acids from lipidperoxidation. Vitamin C has been reported as being capableof regenerating vitamin E. [34]. As vitamin E also plays a rolein free radical attainment, CoQH2 can regenerate vitamin Efrom subsequent α-tocopherol radical formation. In animalmodels, depleted levels of α-tocopherol were followed by thesubsequent oxidation of ubiquinol. *is suggests thatα-tocopherol and ubiquinol act in concert to quench freeradicals [35].

*e synergistic interaction between LA and Q10 wasdemonstrated by Wagner et al. in cultured skeletal musclecells; they found that this combination significantly in-creased nuclear levels of PGC1α, a master switch of energymetabolism and mitochondrial biogenesis. Furthermore,

Table 2: Oxidative stress in the studied groups.

Group I(N� 8)

Group II(N� 7)

Group III(N� 8)

Group IV(N� 8)

Group V(N� 8)

P value ingroup II(cisplatin)versus other

groups

P value in group V(combination)

versusgroups III, IV(monotherapy)

TNF-α(pg/ml)Mean± SD

29.45± 457 53.92± 0.477 47.22± 0.376 47.86± 0.162 46.02± 0.432 0.018∗ NS

MDA content(nmol/g wettissue)Mean± SD

407.21± 13.1 702.43± 33.21 551.5± 9.15 587.75± 13.4 433.25± 9.62 0.026∗ 0.014∗

GSH content(mmol/g wettissue)Mean± SD

0.366± 0.0024 0.1726± 0.0059 0.2179± 0.0081 0.2011± 0.0072 0.2377± 0.0049 0.0167∗ NS

SOD (U/gwet tissue)Mean± SD

7.57± 0.1077 4.16± 0.211 6.41± 0.1016 6.68± 0.055 6.18± 0.0529 0.009∗ NS

CAT (U/gwet tissue)Mean± SD

0.595± 0.0041 0.4653± 0.0199 0.5244± 0.0063 0.5343± 0.0057 0.5728± 0.0079 0.011∗ NS

TNF: tumor necrosis factor. SOD: superoxide dismutase. MDA: malondialdehyde. GSH: glutathione content of renal tissue. CAT: catalase enzyme.∗Significant. NS: nonsignificant.

GIGIIGIII

GIVGV

407.2

702.43∗

551.5 587.75

433.25∗∗

0

100

200

300

400

500

600

700

800

MDA content

Figure 3: MDA content in the studied groups. MDA was sig-nificantly higher when group II was compared to other groups. ∗Pvalue was 0.026. Combination therapy resulted in a significantreduction of MDA when compared to monotherapy (i.e., group Vcompared to group III and group IV). ∗∗P value was 0.014.

International Journal of Inflammation 7

(a) (b)

(c) (d)

(e) (f )

Figure 4: (a) Control: kidney showing average renal capsule (black arrow), average glomeruli (blue arrows), average tubules (red arrow),and average interstitium (H&E ×200). (b) Cisplatin: kidney showing partially detached renal capsule (black arrow), scattered atrophicglomeruli (blue arrows), dilated tubules (yellow arrows), and markedly dilated congested interstitial blood vessels (green arrow) (H&E×200). (c) Cisplatin +CoQ10: kidney showing average renal capsule (black arrow), scattered atrophic glomeruli (blue arrows), dilatedtubules (yellow arrows), and dilated congested blood vessel (red arrow) (H&E ×200). (d) Cisplatin +ALA: kidney showing average renalcapsule (black arrow), scattered small-sized glomeruli (blue arrows), and dilated tubules (yellow arrow) (H&E ×200). (e)Cisplatin +CoQ10 +ALA: kidney showing average renal capsule (black arrow), average glomeruli (blue arrows), and mildly congestedinterstitial blood vessels (yellow arrow) (H&E+ 200). (f ) Cisplatin +CoQ10 +ALA: high power view showing average glomeruli (G) withaverage Bowman’s spaces (BS), proximal tubules (P) with edematous epithelial lining (black arrows) and preserved brush borders, andintratubular hyaline casts (blue arrows) (H&E ×400).

Table 3: Histopathological results.

Renal capsule Glomeruli BS Tubular lining Tubular lumen Brush border Interstitium MedullaGroup I 0 0 0 0 0 0 0 0Group II + ++ + ++ ++ ++ ++ ++Group III 0 ++ + + ++ + ++ +Group IV 0 + + + ++ + + +Group V 0 0 0 + ++ + + +Renal capsule: 0: average +: partially detached ++: completely detached. Glomeruli (G): 0: average +: small-sized/congested ++: atrophied. Bowman’s spaces(BS): 0: average + : widened/dilated ++: obliterated. Tubular lining: 0: average +: edematous/apoptotic ++: necrotic. Tubular lumen: 0: free +: cellular debris++: hyaline casts. Brush border: 0: preserved +: partial loss ++: complete loss. Interstitium: 0: average +: mildly dilated BV ++: markedly dilated BV. Medulla:0: average +: cellular debris/ hyaline casts ++: atrophied lining.

8 International Journal of Inflammation

supplementation with LA plus Q10 resulted in an increase ofgenes encoding proteins involved in stress response, GSHsynthesis, and its recycling. In ALA-plus-Q10-treatedmuscle cells, a significant 4-fold increase in GSH was evi-dent. *is increase in GSH was accompanied by increasednuclear Nrf2 (nuclear factor erythroid-like 2 derived factor2) protein levels, partly regulating cGCS (c-gluta-mylcysteine-synthetase) and GST (glutathione-S-transfer-ases) gene expression. *ese led to the improvement ofenergy homeostasis, stress response, and antioxidant defensemechanisms. Treatment with combined therapy resulted inthe elevation of glutathione levels to higher levels comparedto monotherapy [36].

In our study, the difference among groups treated withmonotherapy versus combined therapy was not significantin all aspects; however, there are two significant findings infavor of combined therapy. *e first is the improved cre-atinine clearance in the group treated with combinedtreatment (creatinine clearance is an important marker forrenal function). *e second is the significant decrease in theMDA level among the combination group (MDA is animportant marker for lipid peroxidation); this reflects theeffectiveness of the combined therapy in reducing the oxi-dative stress mediated by cisplatin. A third notable findingthat supports combined therapy is the less kidney damagedetected by histopathology when compared with each of themonotherapy groups.

Lipid peroxidation involves the loss of hydrogen from apolyunsaturated fatty acid, resulting in a peroxyl radical.Reduced CoQH2 (ubiquinol) loosely holds electrons andacts to eliminate lipid peroxyl radicals by either directlyproducing semiquinone radical (CoQH·) or scavengingother peroxyl radicals [36]. In our study, there was asignificant reduction of MDA level when CoQ10 or ALAwas used. Moreover, the combined therapy was moresignificant than monotherapy in reducing MDA and hencethe oxidative stress. Similar results were given byAhmadvand et al. while examining the possible protectiveeffect of CoQ10 in vivo and in vitro regarding lipid per-oxidation, antioxidant enzyme activity, and glomerulo-sclerosis in alloxan-induced type 1 diabetic rats. *eyfound that coenzyme Q10 significantly antagonizes theelevated levels of MDA in serum and kidney in the treatedgroup compared with the diabetic untreated group. *eyconcluded that CoQ10 exerts beneficial effects on the lipidperoxidation and antioxidant enzyme activity in alloxan-induced type 1 diabetic rats [37].

*e effect of monotherapy with CoQ10 in diminishingrenal injury mediated by cisplatin was investigated by Fouadet al. In that study CoQ10 treatment was started one daybefore cisplatin injection and continued for 6 consecutivedays. CoQ10 caused a significant reduction in blood ureanitrogen and serum creatinine levels, which were increasedby cisplatin. CoQ10 significantly compensated deficits in theantioxidant defense mechanisms (reduced GSH level andSOD activity), suppressed lipid peroxidation, and decreasedthe elevations of TNF-α. Also, histopathological renal tissuedamage mediated by cisplatin was ameliorated by coenzymeQ10 treatment [38]. *e results of that study are comparable

to the results of our study even though CoQ10 was given for6 days after cisplatin injection in the other study and wasgiven for 3 days only in our study. *is may support the ideathat giving a pretreatment course (9 days in our study) isbeneficial and comparable with the posttreatment course.

*e role of ALA in preventing kidney injury was con-firmed in different studies. *is led us to combine ALA withCoQ10 in the current study to maximize the potentialtherapeutic benefits. An example of these studies is the oneconducted by Pianta et al. *ey demonstrated that ALAplays an important role in the amelioration of cisplatin-induced acute kidney injury.*ey found that ALA treatmentattenuated renal tubular injury scores (P< 0.05) and de-creased serum creatinine and urinary damage biomarkers ofproximal tubular injury. Protection was demonstrated byreduced structural damage, improved glomerular filtration,and reduced excretion of urinary biomarkers of proximaltubular damage [39]. *ese data are comparable to our datain the groups treated with ALA; however, the renal damagein our study may be objectively higher since ALA was givenmainly as prophylactic therapy and continued only for 3days after cisplatin exposure; on the other hand, the his-tological findings were almost comparable when the com-bination treatment was given.

Another study by Jamor et al. investigated the impact ofALA against kidney injury in alloxan-induced diabetic rats.ALA (100mg/kg) was given intraperitoneally (IP), daily to 6weeks. Treatment with ALA led to a significant elevation incatalase and GSH levels with a reduction in MDA levels.Histopathological lesions such as increased glomerularvolume and lymphocyte infiltration were attenuated in theALA treated group. *ey concluded that ALA supplemen-tation is effective in preventing complications induced byoxidative stress and atherosclerosis in diabetic rats [40]. Incontrast to our study, ALA was given for 6 weeks and thisresulted in a better histopathological outcome.

It is worth mentioning that ALA has different modes ofaction that contribute better to the alleviation of the toxicdamage in the kidney caused by cisplatin or other com-pounds having heavy metals in their structure. For example,the protective effect of ALA has been tested against cad-mium-induced nephrotoxicity by Luo et al. *eir findingsindicated that the antioxidant, cadmium chelation, andantiapoptotic activities of ALA are the key factors that al-leviate nephrotoxicity [41]. De Sousa Alegre et al. reportedthat ALA alone increases urinary osmolality, decreasesurinary urea and creatinine, and diminishes urinary contentof uric acid [17].

Our study design was focused on the prophylactic effectof both ALA and CoQ10, rather than treatment of kidneyinjury; thus, the treatment was given 9 days before the in-jection of cisplatin, and the samples were taken 72 hoursafter cisplatin administration. In most of the above-mentioned studies, concomitant administration of cisplatinand the tested agent were applied; however, our data are ingeneral comparable to these studies. Adding ALA to CoQ10in the current study improved the outcome of our proposedprophylactic course. *is raises the idea that giving a pre-treatment course along with posttreatment course may

International Journal of Inflammation 9

result in possibly better outcome; this point needs furtherstudy and investigation.

5. Conclusion

*e current study demonstrated the beneficial cumulativeeffect of combining CoQ10 and ALA in reducing the toxicdamage caused by cisplatin to the kidney. More studies areneeded to compare the results of this pretreatment com-bined regimen with a posttreatment regimen.

Data Availability

*e original data used to support the findings of this studyare available from the corresponding author upon request.

Conflicts of Interest

*e authors declare that they have no conflicts of interest.

References

[1] R. P. Miller, R. K. Tadagavadi, G. Ramesh, and W. B. Reeves,“Mechanisms of cisplatin nephrotoxicity,” Toxins, vol. 2,no. 11, pp. 2490–2518, 2010.

[2] L. H. Einhorn, “Curing metastatic testicular cancer,” Pro-ceedings of the National Academy of Sciences, vol. 99, no. 7,pp. 4592–4595, 2002.

[3] T. Karasawa and P. S. Steyger, “An integrated view of cis-platin-induced nephrotoxicity and ototoxicity,” ToxicologyLetters, vol. 237, no. 3, pp. 219–227, 2015.

[4] R. Skinner, A. Pearson, M. English et al., “Cisplatin dose rateas a risk factor for nephrotoxicity in children,” British Journalof Cancer, vol. 77, no. 10, pp. 1677–1682, 1998.

[5] M. Kuhlmann, G. Burkhardt, and H. Kohler, “Insights intopotential cellular mechanisms of cisplatin nephrotoxicity andtheir clinical application,” Nephrology Dialysis Transplanta-tion, vol. 12, no. 12, pp. 2478–2480, 1997.

[6] D. Khynriam and S. B. Prasad, “Changes in glutathione-re-lated enzymes in tumor-bearing mice after cisplatin treat-ment,” Cell Biology and Toxicology, vol. 18, no. 6, pp. 349–358,2002.

[7] P. Randjelovic, S. Veljkovic, and N. Stojiljkovic, “Gentamycinnephrotoxicity in animals: current knowledge and futureperspectives,” EXCLI Journal, vol. 16, pp. 388–399, 2017.

[8] S. Faubel, E. C. Lewis, L. Reznikov et al., “Cisplatin-inducedacute renal failure is associated with an increase in the cy-tokines interleukin (IL)-1β, IL-18, IL-6, and neutrophil in-filtration in the kidney,” Journal of Pharmacology andExperimental Derapeutics, vol. 322, no. 1, pp. 8–15, 2007.

[9] S. Camano, A. Lazaro, E. Moreno-Gordaliza et al., “Cilastatinattenuates cisplatin-induced proximal tubular cell damage,”Journal of Pharmacology and Experimental Derapeutics,vol. 334, no. 2, pp. 419–429, 2010.

[10] K. J. Cullen, Z. Yang, L. Schumaker, and Z. Guo, “Mito-chondria as a critical target of the chemotheraputic agentcisplatin in head and neck cancer,” Journal of Bioenergeticsand Biomembranes, vol. 39, no. 1, pp. 43–50, 2007.

[11] M. Irshad and P. S. Chaudhari, “Oxidant and antioxidantsystem: role and significance in human body,” Indian Journalof Experimental Biology, vol. 40, pp. 1233–1239, 2002.

[12] J. Feher, E. Nemeth, V. L. Nagy, and G. Lengyel, “*e pre-ventive role of coenzyme Q10 and other antioxidants in

injuries caused by oxidative stress,” Archives of Medical Sci-ence, vol. 3, no. 4, pp. 305–314, 2007.

[13] L. Packer, K. Kraemer, and G. Rimbach, “Molecular aspects oflipoic acid in the prevention of diabetes complications,”Nutrition, vol. 17, no. 10, pp. 888–895, 2001.

[14] F. A. Moura, K. Q. Andrade, J. C. Santo, and M. F. Goulart,“Lipoic acid: its antioxidant and anti-inflammatory role andclinical applications,” Current Topics in Medicinal Chemistry,vol. 15, no. 5, 2015.

[15] G. G. Mackenzie, M. P. Zago, A. G. Erlejman, C. L. Keen,L. Aimo, and P. I. Oteiza, “α-lipoic acid and N-acetyl cysteineprevent zinc deficiency-induced activation of NF-κB and AP-1 transcription factor in human neuroblastoma IMR-32 cells,”Free Radical Research, vol. 40, no. 1, pp. 75–84, 2006.

[16] D. Tibullo, G. Li Volti, C. Giallongo et al., “Biochemical andclinical relevance of alpha lipoic acid: antioxidant and anti-inflammatory activity, molecular pathways and therapeuticpotential,” Inflammation Research, vol. 66, no. 11,pp. 947–959, 2017.

[17] V. de Sousa Alegre, J. M. Barone, S. C. Yamasaki, L. Zambotti-Villela, and P. F. Silveira, “Lipoic acid effects on renalfunction, aminopeptidase activities and oxidative stress inCrotalus durissus terrificus envenomation in mice,” Toxicon,vol. 56, no. 3, pp. 402–410, 2010.

[18] K. Winiarska, D. Malinska, K. Szymanski, M. Dudziak, andJ. Bryla, “Lipoic acid ameliorates oxidative stress and renalinjury in alloxan diabetic rabbits,” Biochimie, vol. 90, no. 3,pp. 450–459, 2008.

[19] G. P. Littarru and L. Tiano, “Bioenergetic and antioxidantproperties of coenzyme Q10: recent developments,” Molec-ular Biotechnology, vol. 37, no. 1, pp. 31–37, 2007.

[20] N. Bhaghvan and R. K. Chopra, “Coenzyme Q10: absorption,tissue uptake, metabolism and Pharmacokinetics,” FreeRadical Research, vol. 40, no. 5, pp. 445–453, 2006.

[21] J. Lance, S. McCabe, R. L. Clancy, and J. Pierce, “CoenzymeQ10—a therapeutic agent,” MEDSURG Nursing, vol. 21,pp. 367–371, 2012.

[22] M. Alcazar-Fabra, P. Navas, and G. Brea-Calvo, “Coenzyme Qbiosynthesis and its role in the respiratory chain structure,”Biochimica et Biophysica Acta (BBA)—Bioenergetics, vol. 1857,no. 8, pp. 1073–1078, 2016, þ.

[23] Y.-T. Kuo, P.-H. Shih, S.-H. Kao, G.-C. Yeh, and H. M. Lee,“Pyrroloquinoline quinone resists denervation-inducedskeletal muscle atrophy by activating PGC-1α and integratingmitochondrial electron transport chain complexes,” PLoSOne, vol. 10, no. 12, þ, 2015.

[24] A. W. Linnane and H. Eastwood, “Cellular redox poisemodulation: the role of co enzyme Q10, gene and metabolicregulation,”Mitochondrion, vol. 4, no. 5-6, pp. 779–789, 2004.

[25] Y. Nozaki, K. Kinoshita, S. Hino et al., “Signaling Rho-kinasemediates inflammation and apoptosis in T cells and renaltubules in cisplatin nephrotoxicity,” American Journal ofPhysiology-Renal Physiology, vol. 308, no. 8, pp. F899–F909,2015.

[26] H. S. El-Abhar, “Coenzyme Q10: a novel gastroprotectiveeffect via modulation of vascular permeability, prostaglandinE2, nitric oxide and redox status in indomethacin-inducedgastric ulcer model,” European Journal of Pharmacology,vol. 649, no. 1–3, pp. 314–319, 2010.

[27] R. H. Abdou and M. M. Abdel-Daim, “Alpha-lipoic acidimproves acute deltamethrin-induced toxicity in rats,” Ca-nadian Journal of Physiology and Pharmacology, vol. 92, no. 9,pp. 773–779, 2014.

10 International Journal of Inflammation

[28] G. L. Ellman, “Tissue sulfhydryl groups,” Archives of Bio-chemistry and Biophysics, vol. 82, no. 1, pp. 70–77, 1959.

[29] S. Marklund, “Pyrogallol autooxidation,” in Handbook ofMethods for Oxygen Radical Research 1985, pp. 243–247, CRCPress, Boca Raton, FL, USA, 1985.

[30] A. Claiborne, Catalase Activity; Handbook of Methods for Ox-ygen Radical Research, CRC Press, Boca Raton, FL, USA, 1985.

[31] M. Mihara and M. Uchiyama, “Determination of malo-naldehyde precursor in tissues by thiobarbituric acid test,”Analytical Biochemistry, vol. 86, no. 86, pp. 271–278, 1978.

[32] P. Martina and V. H. Zeljka, “Cispaltin- induced rodentmodel of kidney injury; characteristics and challenges,”BioMed Research International, vol. 2018, Article ID 1462802,29 pages, 2018.

[33] L. Badimon, T. G. Vilahur, and T. Padro, “Systems biologyapproaches to understand the effects of nutrition and promotehealth,” British Journal of Clinical Pharmacology, vol. 83, no. 1,pp. 38–45, 2017.

[34] E. Cadenas, L. Packer, and M. G. Traber, “Antioxidants,oxidants, and redox impacts on cell function -a tribute toHelmut Sies-,” Archives of Biochemistry and Biophysics,vol. 595, pp. 94–99, 2016, þ.

[35] H. A. Peres, M. C. Foss, and L. R. Pereira, “*e role of co-enzyme Q10 supplementation with statin drug use andchronic diseases,” Journal of Ancient Diseases & PreventiveRemedies, vol. 5, no. 2, pp. 1–3, 2017.

[36] A. E. Wagner, M. A. Ernst, M. Birringer, O. Sancak, L. Barella,and G. Rimbach, “A combination of lipoic acid plus coenzymeQ10 induces PGC1α, a master switch of energy metabolism,improves stress response, and increases cellular glutathionelevels in cultured C2C12 skeletal muscle cells,” OxidativeMedicine and Cellular Longevity, vol. 2012, Article ID 835970,9 pages, 2012.

[37] H. Ahmadvand, M. Tavafi, and A. Khosrowbeygi, “Amelio-ration of altered antioxidant enzymes activity and glomer-ulosclerosis by coenzyme Q10 in alloxan-induced diabeticrats,” Journal of Diabetes and Its Complications, vol. 26, no. 6,pp. 476–482, 2012.

[38] A. A. Fouad, A. I. Al-Sultan, S. M. Refaie, and M. T. Yacoubi,“Coenzyme Q10 treatment ameliorates acute cisplatinnephrotoxicity in mice,” Toxicology, vol. 274, no. 1–3,pp. 49–56, 2010, þ.

[39] T. J. Pianta, L. Succar, T. Davidson, N. A. Buckley, andZ. H. Endre, “Monitoring treatment of acute kidney injurywith damage biomarkers,” Toxicology Letters, vol. 268,pp. 63–70, 2017.

[40] P. Jamor, H. Ahmadvand, H. Ashoory, and E. Babaeenezhad,“Effect of alpha-lipoic acid on antioxidant gene expressionand kidney injury in alloxan-induced diabetic rats,” Journal ofNephropathology, vol. 8, no. 1, 2019.

[41] T. Luo, G. Liu, M. Long et al., “Treatment of cadmium-in-duced renal oxidative damage in rats by administration ofalpha-lipoic acid,” Environmental Science and Pollution Re-search, vol. 24, no. 2, pp. 1832–1844, 2017.

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